U.S. patent application number 11/136841 was filed with the patent office on 2006-02-09 for chips for multiplex analyses.
Invention is credited to Amjad Huda, Victor Joseph, Alnoor Shivji, Jie Zhou.
Application Number | 20060030036 11/136841 |
Document ID | / |
Family ID | 35463443 |
Filed Date | 2006-02-09 |
United States Patent
Application |
20060030036 |
Kind Code |
A1 |
Joseph; Victor ; et
al. |
February 9, 2006 |
Chips for multiplex analyses
Abstract
The present invention provides miniaturized instruments for
conducting chemical reactions where control of the reaction
temperature is desired or required. Specifically, this invention
provides chips and optical systems for performing and monitoring
temperature-dependent chemical reactions. The apparatus and methods
embodied in the present invention are particularly useful for
high-throughput and low-cost amplification of nucleic acids.
Inventors: |
Joseph; Victor; (Fremont,
CA) ; Huda; Amjad; (Fremont, CA) ; Shivji;
Alnoor; (Fremont, CA) ; Zhou; Jie; (Mason,
OH) |
Correspondence
Address: |
WILSON SONSINI GOODRICH & ROSATI
650 PAGE MILL ROAD
PALO ALTO
CA
94304-1050
US
|
Family ID: |
35463443 |
Appl. No.: |
11/136841 |
Filed: |
May 24, 2005 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10857552 |
May 28, 2004 |
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11136841 |
May 24, 2005 |
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60630789 |
Nov 24, 2004 |
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Current U.S.
Class: |
435/288.4 ;
435/288.7; 435/303.1 |
Current CPC
Class: |
B01L 2300/0829 20130101;
B01L 2300/18 20130101; G01N 21/6452 20130101; B01L 2300/044
20130101; G01N 2201/0231 20130101; H01L 31/02325 20130101; C12Q
1/6806 20130101; B01L 2200/0689 20130101; H01L 31/0203 20130101;
G01N 21/76 20130101; G01N 2035/0405 20130101; B01L 2300/1827
20130101; B82Y 30/00 20130101; H01L 31/024 20130101; B01L 2300/0654
20130101; C12Q 1/6844 20130101; B01L 3/50853 20130101; B01L 7/52
20130101; B01L 3/50851 20130101; H01L 27/1443 20130101; G01N
2021/6484 20130101; G01N 21/6408 20130101; B01L 7/54 20130101; C12Q
1/686 20130101; G01N 35/0099 20130101; H01L 31/105 20130101; G01N
2021/6417 20130101; B01L 2300/0819 20130101; G01N 21/01 20130101;
H01L 31/18 20130101; B01L 2300/0636 20130101; B01L 2300/1822
20130101; G01N 21/0332 20130101; C12Q 1/6844 20130101; C12Q
2565/501 20130101 |
Class at
Publication: |
435/288.4 ;
435/288.7; 435/303.1 |
International
Class: |
C12M 1/34 20060101
C12M001/34 |
Claims
1. A chip comprising an indium tin oxide heater (ITO-heater) in a
glass plate that is coupled to an array of thermo-controllable
units fabricated in the chip.
2. The chip of claim 1, wherein the individual unit within the
array comprises a micro well for receiving and confining the
reaction sample.
3. The chip of claim 2, wherein the micro well is sealed by (a)
applying a radiation-curable adhesive along peripheral dimensions
defining an open surface of the micro well; (b) placing a cover to
encompass the peripheral dimensions that define the open surface of
the micro well; and (c) exposing the micro well to a radiation beam
to effect the sealing.
4. The chip of claim 3, wherein the radiation-curable adhesive is
UV-curable adhesive.
5. The chip of claim 2, wherein the well has a dimension of about
10 mm to about 100 .mu.m in length, about 100 mm to about 100 .mu.m
in width, and about 10 mm to about 100 .mu.m in depth.
6. The chip of claim 2, wherein the micro well has a volume of from
about 100 .mu.l to about 0.001 .mu.l.
7. The chip of claim 2, wherein the well has an upper and a bottom
surface, and wherein the indium tin oxide heater in a glass plate
is attached to the bottom surface or the upper surface but not
both.
8. The chip of claim 2, wherein the well has an upper and a bottom
surface, and wherein the wherein the indium tin oxide heater in a
glass plate is attached to both the upper and the bottom
surfaces
9. The chip of claim 1, wherein the chip comprises a photon-sensing
element.
10. The chip of claim 1, wherein the chip is operatively coupled to
an optical system that detects optical signals emitted from the
reaction sample.
11. The chip of claim 1, wherein the chip has a ramp temperature
time about 25.degree. C. or higher per second.
12. The chip of claim 2, wherein each unit further comprises a
temperature sensor in thermal contact with the micro well.
13. The chip of claim 2, wherein the well has an upper and a bottom
surface, said upper surface having a temperature higher than that
of the bottom surface.
14. An apparatus for conducting a chemical reaction requiring
cycling at least two temperature levels, comprising: (a) a chip for
cycling at least two temperature levels, said array comprising: an
array of thermo-controllable units, said array being in thermal
contact with a heating element comprising an indium tin oxide
heater (ITO-heater) in a glass plate, wherein individual unit
within the array comprises a micro well for receiving and confining
a reaction sample, said well being sealed upon filling the reaction
sample; (b) an optical system operatively coupled to the chip of
(a), wherein the optical system detects an optical signal coming
from the micro well.
15. The apparatus of claim 14, wherein the micro well is sealed by
(a) applying a radiation-curable adhesive along peripheral
dimensions defining an open surface of the micro well; (b) placing
a cover to encompass the peripheral dimensions that define the open
surface of the micro well; and (c) exposing the micro well to a
radiation beam to effect the sealing.
16. The apparatus of claim 14, wherein the chemical reaction is
nucleic acid amplification reaction.
17. A method of conducting a chemical reaction that involves a
plurality of reaction samples and requires cycling at least two
temperature levels, comprising: (a) providing a chip comprising: an
array of thermo-controllable units, said array being in thermal
contact with a heating element comprising an indium tin oxide
heater (ITO-heater) in a glass plate, wherein individual unit
within the array comprises a micro well for receiving and confining
a reaction sample, said well being sealed upon filling the reaction
sample; (b) placing the plurality of reaction samples into the
thermo-controllable units; and (c) controlling the heating element
to effect cycling at least two temperature levels.
18. The method of claim 17, wherein the controlling step comprises
processing sensor signals retrieved from a temperature sensor
operatively linked to a thermo-controllable unit based on protocol
stored on a computer readable medium.
19. The method of claim 17, wherein the chip is operatively coupled
to an optical system that detects optical signals emitted from the
reaction sample.
20. The method of claim 17, wherein the optical signals are
proportional to the amount of product of the chemical reaction.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims the priority benefit of U.S.
Provisional Application Ser. No. 60/630,789 filed Nov. 24, 2004,
and U.S. patent application Ser. No. 10/857,552 filed May 28, 2004,
all of which are hereby incorporated herein by reference in their
entirety.
TECHNICAL FIELD
[0002] This invention relates to miniaturized instruments for
conducting chemical reactions where control of the reaction
temperature is desired or required. Specifically, the invention
relates to chips and optical systems for performing
temperature-dependent chemical reactions. The apparatus and methods
embodied in the present invention are particularly useful for
high-throughput and low-cost amplification of nucleic acids.
BACKGROUND OF THE INVENTION
[0003] The advent of Polymerase Chain Reaction (PCR) since 1983 has
revolutionized molecular biology through vastly extending the
capability to identify, manipulate, and reproduce DNA. Nowadays PCR
is routinely practiced in the course of conducting scientific
researches, clinical diagnostics, forensic identifications, and
environmental studies. PCR has been automated through the use of
thermal stable DNA polymerases and a machine commonly known as
"thermal cycler".
[0004] Performing a large quantity of PCR with the conventional
thermal cycler has been rather expensive. This is partly due to the
intrinsic limitations of the conventional instrument. The
conventional thermal cycler employs metal heating blocks and
cooling reservoirs to carry out the thermal cycling of reaction
samples in plastic microfuge tubes. Because the instrument has a
large thermal mass and the plastic tubes have low heat
conductivity, high level of electrical power is required to operate
the instrument.
[0005] In recent years, the advancement in microfabrication
technology enabled the production of miniaturized devices
integrated with electrical, optical, chemical or mechanical
elements. The technology embodies a range of fabrication techniques
including low-pressure vapor deposition, photolithography, and
etching. Based on these techniques, miniaturized devices containing
silicon channels coupled to micro-heaters have been proposed (see,
e.g., U.S. Pat. Nos. 5,639,423, 5,589,136 and 5,587,128). While the
channel- or chamber-based design in principle reduces the thermal
mass and the reaction volume, it still suffers from other practical
drawbacks. In particular, the channels or chambers by design are
not amenable to automated sealing that prevents evaporation and/or
cross contamination of the reaction samples.
[0006] There thus remains a considerable need for small, mass
produced, and disposable devices designed to perform
high-throughput amplification of nucleic acids. A desirable device
would allow (a) multiplexing an enormous quantity of amplification
reactions; (b) automated and targeted sealing of the reaction sites
on the devices; and (c) monitoring the progress of the
amplification reaction in real time. The present invention
satisfies these needs and provides related advantages as well.
SUMMARY OF THE INVENTION
[0007] A principal aspect of the present invention is the design of
miniaturized devices applicable for multiplex analyses of
individual molecules, and/or simultaneous performance of a vast
number of chemical reactions. The devices and methods of the
present invention simplify the laborious and expensive procedures
of performing chemical reactions where a control of the reaction
temperature is desired or required.
[0008] Accordingly, in one embodiment, the present invention
provides a chip for varying and/or maintaining temperature of a
reaction sample. The chip comprises an array of thermo-controllable
units that is in thermal contact with a heating element, wherein
the varying and/or maintaining of temperature is achieved by
controlling the heating element alone, and wherein individual unit
within the array comprises a micro well for receiving and confining
the reaction sample.
[0009] In another embodiment, the present invention provides a chip
for varying and/or maintaining a reaction sample that comprises an
array of thermo-controllable units, wherein the chip has a surface
density of at least about one thermo-controllable unit per 1
mm.sup.2, and wherein a unit within the array comprises a micro
well for receiving and confining the reaction sample, and a heating
element in thermal contact with the micro well.
[0010] In yet another embodiment, the present invention provides a
chip that comprises two arrays of thermo-controllable units,
wherein one array is arranged in one orientation along the upper
surface, and wherein the other array is arranged in an opposite
orientation along the bottom surface.
[0011] In a further embodiment, the present invention provides a
chip comprising an indium tin oxide heater (ITO-heater) in a glass
plate that is coupled to an array of thermo-controllable units
fabricated in the chip.
[0012] In one aspect of these embodiments, the micro well is sealed
upon filling the reaction sample. Preferably, the well is sealed by
(a) applying a radiation-curable adhesive along peripheral
dimensions defining an open surface of the micro well; (b) placing
a cover to encompass the peripheral dimensions that define the open
surface of the micro well; and (c) exposing the micro well to a
radiation beam to effect the sealing. A wide range of radiation
curable adhesive is applicable for the present invention. They
include but are not limited to a diversity of acrylics, acrylates,
polyurethanes (PUR), polyesters, vinyl, vinyl esters, and epoxies
that are curable by radiation beams such as UV radiation and other
radiation beams of various frequencies.
[0013] In certain aspects, the micro well in the subject chips are
about 10 mm to about 100 .mu.m in length, about 10 mm to about 100
.mu.m in width, and about 10 mm to about 100 .mu./m in depth. The
volume of the micro well is generally small, ranging from about
0.001 .mu.l to about 100 .mu.l. Where desired, not all of the
thermo-controllable units within an array are filled with reaction
sample. Preferably, any two thermo-controllable units filled with a
reaction sample are separated by at least one unfilled
thermo-controllable unit. In certain aspects, the subject chips
have a surface density of at least one thermo-controllable unit per
cm.sup.2, preferably at least about 10 per cm.sup.2, or preferably
at least about 100 per cm.sup.2, or preferably in the range of
about 5 thermo-controllable units to about 50 thermo-controllable
units per mm.sup.2. Where desired, the chips are operatively linked
to a dispensing system for placing a reaction sample into the
thermo-controllable units.
[0014] In other aspects, the thermo-controllable units of the
subject chips can be arranged in different temperature zones, each
of which can be separately controlled. In general, the micro well
within each unit is in thermal contact with a heating element that
can be made of metal or semi-conducting material. Preferred heating
element is an indium-tin-oxide (ITO) heater. Heating element can be
located within the micro well, or can be attached to the upper or
bottom, or both surfaces of the micro well. To prevent evaporation
and condensation of the reaction sample on the upper surface of the
well, the upper surface can be maintained at a higher temperature
than the bottom surface. In preferred embodiments, a plurality of
grooves is fabricated to the bottom surface of the chip. The
presence of the grooves greatly facilitates passive heat
dissipation during thermal cycling. In other embodiments, the
subject chips have a ramp temperature time about 10.degree. C. or
higher per second, preferably about 25.degree. C. or higher per
second. In other preferred embodiments, the subject chips may
comprise temperature sensors in thermal contact with the micro
wells.
[0015] In certain embodiments, the subject chips are operatively
coupled to an optical system that detects optical signals emitted
from the reaction sample. In preferred embodiments, the subject
chips are fabricated with photon-sensing elements in optical
communication with the micro wells where chemical reactions are
taking place. Representative photon-sensing elements include photo
multiplier tube, charge coupled device, avalanche photo diode, gate
sensitive FET's and nano-tube FET's, and P-I-N diode.
[0016] The present invention also provides an apparatus for
conducting a chemical reaction requiring cycling at least two
temperature levels. The apparatus comprises a chip of the present
invention and an optical system that is operatively coupled to the
chip. In this apparatus, the optical system detects an optical
signal such as luminescent signal coming from the micro well
fabricated in the chip. In one aspect, the optical system monitors
the optical signal over a multiple-cycle period. In another aspect,
the optical system detects an optical signal that is proportional
to the amount of product of the chemical reaction taking place in
the micro well over a multiple-cycle period. The optical system can
include a spectrum analyzer that is composed of an optical
transmission element and a photon-sensing element. Preferred
optical transmission element can be selected from the group
consisting of multi-mode fibers (MMF), single-mode fibers (SMF) and
a waveguide. Preferred photon-sensing element can be selected from
the group consisting of photo multiplier tube, charge coupled
device, avalanche photo diode, gate sensitive FET's and nano-tube
FET's, and P-I-N diode.
[0017] In a preferred embodiment, the present invention provides an
apparatus for multiplexed analysis. The apparatus comprises an
array of micro wells for containing and confining reaction samples,
wherein the array is optically linked to (a) an optical multiplexer
adapted for receiving and multiplexing a plurality of incoming
beams of one or more different wavelengths; (b) an optical switch
adapted for redirecting the multiplexed wavelengths of (a) to
individual output fibers, wherein each of the individual output
fibers optically linked to a micro well of the array, said micro
well being coupled to a spectrum analyzer that spectrally analyzes
optical signals coming from the micro well.
[0018] The apparatus of the present invention is capable of
performing a vast diversity of chemical reactions. The subject
apparatus is particularly suited for performing enzymatic
reactions, including but not limited to nucleic acid amplification
reaction that encompasses PCR, quantitative polymerase chain
reaction (qPCR), nucleic acid sequencing, ligase chain polymerase
chain reaction (LCR-PCR), reverse transcription PCR reaction
(RT-PCR), reverse transcription, and nucleic acid ligation.
[0019] Also provided by the present invention is a method of
varying and/or maintaining a temperature of a reaction sample. The
method involves (a) placing the reaction sample into a micro well
fabricated in a chip, said micro well being in thermal contact with
a heating element, wherein the varying and/or maintaining of
temperature is achieved by controlling the heating element alone,
and wherein the micro well receives and confines the reaction
sample, and is sealed when filled with the reaction sample; (b)
applying a voltage to the heating element thereby varying and/or
maintaining the temperature of the reaction sample. The step of
applying a voltage can be effected by processing a predetermined
algorithm stored on a computer readable media operatively linked to
the heating element. The method may also involve cycling the
applying step to raise and lower the temperature of the reaction
sample.
[0020] Further provided is a method of using a chip of the present
invention to conduct a chemical reaction that involves a plurality
of reaction samples and requires cycling at least two temperature
levels. The method comprises: (a) providing a subject chip; (b)
placing the plurality of reaction samples into the
thermo-controllable units; and (c) controlling the heating element
to effect cycling at least two temperature levels. In one aspect of
this embodiment, the controlling step comprises processing sensor
signals retrieved from a temperature sensor operatively linked to a
thermo-controllable unit based on protocol stored on a computer
readable medium. In another aspect, the method employs a subject
chip operatively coupled to an optical system that detects optical
signals emitted from the reaction sample. In a preferred aspect,
the optical system detects the optical signal without the need for
opening the micro well once the chemical reaction is initiated. In
yet another preferred aspect, the optical signals detected are
proportional to the amount of product of the chemical reaction. A
variety of chemical reactions including a specific protein-protein
interaction and enzymatic reactions can be performed using this
method.
[0021] The present invention also provides a method of using the
subject apparatus comprising the chip and optical system described
herein to detect the presence or absence of a target nucleic acid
in a plurality of reaction samples. In this method, the formation
of amplified target nucleic acids indicates the presence of the
target nucleic acid in the reaction sample. In certain aspects, the
amplified target nucleic acids are observed by transmitting
excitation beams into the wells containing the reaction samples,
and detecting the optical signals coming from the micro well. In
other aspects, formation of amplified target nucleic acids is
observed by transmitting excitation beams into the wells containing
the reaction samples at a plurality of times during the
amplification, and monitoring the optical signals coming from the
micro well at each of the plurality of times.
BRIEF DESCRIPTION OF THE DRAWINGS
[0022] FIG. 1 depicts one illustrative chip design 101 having an
integrated heating element and photon-sensing element.
[0023] FIG. 2 is a top view of one exemplary chip layout 101 on a
circular substrate 201.
[0024] FIG. 3 is a schematic representation of one micro well on
the chip.
[0025] FIG. 4 is a graph plotting the amount of time required to
lose 10% reaction sample volume (y-axis) at various temperatures
.alpha.-axis). Three different initial droplet volumes, namely 20
nl, 50 nl, and 100 nl are shown. The dew point under the test
condition is about 14.degree. C.
[0026] FIG. 5 depicts one illustrative chip design 501 having a
sealed micro well. The micro well is sealed by first applying a
UV-curable epoxy along the peripheral dimensions that defines the
open surface of the micro well, followed by laying a photo mask
that allows penetration of UV light along the peripheral
dimensions, and then curing the epoxy using the UV light.
[0027] FIG. 6A is a schematic longitudinal cross sectional view of
an exemplary chip design according to the present invention. The
chip comprises several layers of materials, including a substrate
layer fabricated therein a temperature sensor 604, a first heater
606, a photon-sensing device P-I-N diode 608. A waveguide 610, and
a second heater are fabricated in the upper layers. The top layer
comprises etched-in grooves for placement of epoxy for purpose of
sealing the micro well. FIG. 6B depicts an exemplary cover placed
on top of the exemplary chip shown in 6A.
[0028] FIG. 7 is a schematic diagram showing different components
of the overall analysis system.
[0029] FIG. 8 is a top view of one exemplary chip design 1001.
Shown in the figure are 4 thermo-controllable units, each being
surrounded at the base by four L-shaped grooves 1002 (dashed
lines). Each unit contains an integrated heating element 1006.
[0030] FIG. 9 depicts a side view of the chip design of FIG. 8.
[0031] FIG. 10 is a bottom view of the chip design shown in FIG.
8.
[0032] FIG. 11 is a schematic longitudinal cross sectional view
along the cutting line IV shown in FIG. 8.
[0033] FIG. 12 depicts a typical thermal cycling profile using an
exemplary chip described herein.
[0034] FIG. 13 depicts another illustrative chip design of the
present invention. The chip comprises two opposing arrays of
thermo-controllable units. Both arrays can be in thermal contact
with a heater, one being placed on the upper surface, and another
being placed at the bottom surface of the chip.
[0035] FIG. 14 is a top view of an exemplary chip design adopting a
96-well format. Each well is optically linked to an optical
transmission element, multi-mode fibers (MMF).
[0036] FIG. 15 depicts a side view of the chip shown in FIG. 14.
The side view shows a micro well optically linked to an optical
system, having an optical transmission element (e.g., multi-mode
fibers (MMF)) and photon-sensing element (e.g., CCD) on the
top.
[0037] FIG. 16 depicts an apparatus having an array of micro wells
optically linked to an optical system.
[0038] FIG. 17A depicts another apparatus of the present invention
comprising an array of thermo-controllable units optically linked
to an optical system. FIG. 17B depicts a top view of fiber bundle
with localized 8-.lamda. excitation light MMF and single 1 mm MMF
for 8-.lamda. different emission lights in mixed format. This is an
MMF bundled fiber sub-assembly with all central emission MMF fiber
goes through AWG and rest of the excitation to the sides.
[0039] FIG. 18 depicts a 12.times.8 fiber array each having
1.times.8 channel and 18 mm long DE-MUX AWG for passive low loss
emission light separation with real time analysis capability of all
emissions simultaneously from a 16.times.96 well chip. The spectrum
can be resolved and analyzed by EMCCD.
[0040] FIG. 19 depicts another apparatus of the present invention
comprising an array of thermo-controllable units optically linked
to an optical system.
[0041] FIG. 20 depicts the SYBR Green-stained G6PDH gene products
amplified using a chip of the present invention.
[0042] FIG. 21 depicts the SYBR Green staining of G6PDH gene
products appeared at the three different thermal stages of one PCR
cycle. The three stages are primer annealing at 45.degree. C.,
denaturation of DNA at 95.degree. C., and primer-dependent
extension at 72.degree. C.
[0043] FIG. 22 depicts the amount of SYBR stain quantified
throughout one complete thermal cycle.
[0044] FIG. 23 depicts the thermal cycle profiles of a chip of the
present invention. The mean ramp rate is about 28.degree. C. per
second.
[0045] FIG. 24 depicts a chip with more than one temperature
zone.
[0046] FIG. 25 depicts an exemplary method for sealing the subject
micro well.
DETAILED DESCRIPTION OF THE INVENTION
General Techniques:
[0047] The practice of the present invention will employ, unless
otherwise indicated, conventional techniques of Integrated Circuit
(IC) processing biochemistry, chemistry, molecular biology,
genomics and recombinant DNA, which are within the skill of the
art. See, e.g., Stanley Wolf et al., SILICON PROCESSING FOR THE
VLSI ERA, Vols 1-4 (Lattice Press); Michael Quirk et al.,
SEMICONDUCTOR MANUFACTURING TECHNOLOGY; Sambrook, Fritsch and
Maniatis, MOLECULAR CLONING: A LABORATORY MANUAL, 2.sup.nd edition
(1989); the series METHODS IN ENZYMOLOGY (Academic Press, Inc.):
PCR 2: A PRACTICAL APPROACH (M. J. MacPherson, B. D. Hames and G.
R. Taylor eds. (1995).
Definitions:
[0048] As used in the specification and claims, the singular form
"a", "an" and "the" include plural references unless the context
clearly dictates otherwise.
[0049] "Luminescence" is the term commonly used to refer to the
emission of light from a substance for any reason other than a rise
in its temperature. In general, atoms or molecules emit photons of
electromagnetic energy (e.g., light) when then move from an
"excited state" to a lower energy state (usually the ground state);
this process is often referred to as "radioactive decay". There are
many causes of excitation. If exciting cause is a photon, the
luminescence process is referred to as "photoluminescence". If the
exciting cause is an electron, the luminescence process is referred
to as "electroluminescence". More specifically, electroluminescence
results from the direct injection and removal of electrons to form
an electron-hole pair, and subsequent recombination of the
electron-hole pair to emit a photon. Luminescence which results
from a chemical reaction is usually referred to as
"chemiluminescence". Luminescence produced by a living organism is
usually referred to as "bioluminescence". If photoluminescence is
the result of a spin-allowed transition (e.g., a single-singlet
transition, triplet-triplet transition), the photoluminescence
process is usually referred to as "fluorescence". Typically,
fluorescence emissions do not persist after the exciting cause is
removed as a result of short-lived excited states which may rapidly
relax through such spin-allowed transitions. If photoluminescence
is the result of a spin-forbidden transition (e.g., a
triplet-singlet transition), the photoluminescence process is
usually referred to as "phosphorescence". Typically,
phosphorescence emissions persist long after the exciting cause is
removed as a result of long-lived excited states which may relax
only through such spin-forbidden transitions. A "luminescent label"
or "luminescent signal" may have any one of the above-described
properties.
[0050] A "primer" is a short polynucleotide, generally with a free
3'-OH group, that binds to a target nucleic acid (or template)
potentially present in a sample of interest by hybridizing with the
target nucleic acid, and thereafter promoting polymerization of a
polynucleotide complementary to the target.
[0051] The terms "operatively linked to" or "operatively coupled
to" are used interchangeably herein. They refer to a juxtaposition
wherein the components so described are in a relationship
permitting them to function in their intended manner.
Structure of the Chips of the Present Invention
[0052] A central aspect of the present invention is the design of
miniaturized devices applicable for multiplexed analyses of
individual molecules, and/or simultaneous performance of a vast
number of chemical reactions. Distinguished from the previously
reported micro-capillary devices or other channel-based
instruments, the present invention provides a highly automated,
miniaturized, analytical instrument that allows manipulations with
precise control of temperature, evaporation, small-volume reagent
delivery, product detection in a multiplexed fashion.
[0053] In one embodiment, the present invention provides chips for
varying and/or maintaining temperature of a reaction sample. The
chips embodied in the present invention generally comprise at least
one array of thermo-controllable units. The individual units within
the array are separated from each other by a physical barrier
resistant to the passage of liquids. These units generally form
indented areas referred to as "micro wells". A micro well can be
open at the top, but it must be enclosed otherwise and physically
isolated from other wells to restrict passage of liquids.
Accordingly, the micro well has at least one cavity suitable for
receiving and confining reaction sample. By "confining reaction
sample" it is meant that the mobility of the reaction sample is
restricted by a physical barrier, and the sample is prevented from
flowing into another site once it is applied to the micro well of a
thermo-controllable unit.
[0054] The micro well may be fabricated in any convenient size,
shape or volume. The well may be about 10 mm to about 100 .mu.m in
length, about 10 mm to 100 .mu.m in width, and about 10 mm to 100
.mu.m in depth. In general, the well has a transverse sectional
area in the range of about 0.01 mm.sup.2 to about 100 mm.sup.2. The
transverse sectional area may be circular, elliptical, oval,
conical, rectangular, triangular, polyhedral, or in any other
shape. The transverse area at any given depth of the well may also
vary in size and shape.
[0055] The overall size of the chip ranges from a few microns to a
few centimeters in thickness, and from a few millimeters to 50
centimeters in width or length. Preferred chips have an overall
size of about 500 micron in thickness and may have any width or
length depending on the number of micro wells desired.
[0056] The cavity of each well may take a variety of
configurations. For instance, the cavity within a micro well may be
divided by linear or curved walls to form separate but adjacent
compartments, or by circular walls to form inner and outer annular
compartments.
[0057] The micro well typically has a volume of less than 500 ul,
preferably less than 50 ul. The volume may be less than 10 ul, or
even less than 1 ul. Where desired, the micro well can be
fabricated in which the inner surface area to volume ratio is
greater than about 1 mm.sup.2/1 ul, or greater than 5 mm.sup.2/1
ul, or even greater than 10 mm.sup.2/1 ul. Increasing the surface
area to volume ratio facilitates heat transfer through the
thermal-controllable unit, thereby reducing the ramp time of a
thermal cycle.
[0058] A micro well of high inner surface to volume ratio may be
coated with materials to reduce the possibility that the reactants
contained therein may interact with the inner surfaces of the well.
Coating is particularly useful if the reagents are prone to
interact or adhere to the inner surfaces undesirably. Depending on
the properties of the reactants, hydrophobic or hydrophilic
coatings may be selected. A variety of appropriate coating
materials are available in the art. Some of the materials may
covalently adhere to the surface, others may attach to the surface
via non-covalent interactions. Non-limiting examples of coating
materials include silanization reagent such as dimethychlorosilane,
dimethydichlorosilane, hexamethyldisilazane or
trimethylchlorosilane, polymaleimide, and siliconizing reagents
such as silicon oxide, Aquasil.TM., and Surfasil.TM.. Additional
suitable coating materials are blocking agents such as amino acids,
or polymers including but not limited to polyvinylpyrrolidone,
polyadenylic acid and polymaleimide.
[0059] The surface of the micro well can further be altered to
create adsorption sites for reaction reagents. These sites may
comprise linker moieties for attachment of biological or chemical
compound such as a simple or complex organic or inorganic molecule,
a peptide, a protein (e.g. antibody) or a polynucleotide.
[0060] The total number of thermo-controllable units fabricated on
the chip will vary depending on the particular application in which
the subject chips are to be employed. To accommodate the need for
simultaneous performance of a vast number of reactions, the subject
chips will generally comprise at least about 100
thermo-controllable units, usually at least about 500
thermo-controllable units, and more usually at least about 1000
units. The density of the thermo-controllable units on the chip
surface may vary depending on the particular application.
High-density chips usually contains on the surface at least about
10 thermo-controllable units per cm.sup.2, or preferably at least
about 100 thermo-controllable units per cm.sup.2, or preferably in
the range of about 5 thermo-controllable units to about 50
thermo-controllable units per mm.sup.2.
[0061] The thermo-controllable units of the subject chips can be
arrayed in any format across or over the surface of the chip, such
as in rows and columns so as to form a grid, in a circular pattern,
and the like, see, e.g., FIG. 2. In a preferred embodiment, the
thermo-controllable units are arrayed in a format compatible to
instrumentation already exists for dispensing reagents and/or
reading assays on microtiter plates. That way extensive
re-engineering of commercially available fluid handling devices is
not required. Accordingly, the thermo-controllable units of the
subject chips are preferably arranged in an 8.times.12 format.
Using this format, each chip may have at least 96
thermo-controllable units, preferably at least 384
thermo-controllable units, more preferably at least 1,536 units,
and even more preferably 6,144 or 24,576 units. While the number of
thermo-controllable units of the 8.times.12 format chip may be as
many as 24,576 or more, it usually does riot exceed about 393,216
units, and more usually does not exceed about 6,291,456 units.
[0062] The subject chips typically contain one or more grooves
etched in at the bottom side of the chip. In general, the grooves
are under-trenches, open channels or paths to allow air passage.
The grooves reduce the thermal mass of the chip, increase the
surface area, and thus enhance the thermal performance of the
chips. The grooves can be fabricated in any shapes, including but
not limited to circular, elliptical, oval, conical, rectangular,
triangular, and polyhedral. The grooves may be further divided by
linear or curved walls to form separate but adjacent channels, or
by circular walls to form inner and outer annular channels. The
dimensions of the grooves will depend on the overall sizes and
depths of the chips. The depths of the grooves may range from about
one tenth to about nine tenths of the chip depths. The other
dimensions, namely widths and lengths, may be shorter, longer or
comparable to the corresponding dimensions of the chips. FIGS. 8-10
depict an exemplary groove 1002 design. In particular, the L-shaped
grooves surround the base of a thermo-controllable unit. As the air
flows through the passageways formed by any of the grooves, it
removes heat from the surfaces of thermo-controllable unit by
passive heat dissipation, thus increasing the speed of thermal
cycling.
[0063] The subject chips may contain more than one array of
thermo-controllable units. The arrays of thermo-controllable units
may align horizontally, longitudinally, or diagonally long the
x-axes or the y-axes of the chips. In addition, the arrays may
align along curved walls within the chips that divide the arrays to
form separate but adjacent compartments.
[0064] A preferred chip of the present invention comprises at least
two separate arrays of thermo-controllable units. For instance, one
array may be arranged in one orientation along the upper surface of
the chip, and the another array may be arranged in an opposite
orientation along the bottom surface of the chip. By "opposite
orientation" it is meant that one of the arrays is arranged in an
inverted manner so that the top surface of each thermo-controllable
unit of the array (where the reagents can be added prior to sealing
the surface) points away from that of the thermo-controllable units
in the opposing array. The two opposing arrays may be arranged such
that the base of each thermo-controllable unit is directly opposite
to that of the opposing array. Alternatively, the opposing arrays
may be staggered such that the thermo-controllable units of the
opposing arrays are located in between those thermo-controllable
units in the opposing array. FIG. 13 depicts an illustrative chip
comprising at least two opposing arrays. In this Figure, the chip
1301 has an upper 1302 and bottom surface 1304. One of the arrays
is arranged along the upper surface 1306, and the other is arranged
in an opposite orientation along the bottom surface 1308. The
thermo-controllable units of the bottom array are positioned in an
inverted manner so that the open surface of each unit points away
from that of the opposing unit in the chip. The two arrays are
staggered so that a cross-section line VII cuts through the
thermo-controllable units from both the upper and the bottom
arrays.
[0065] Though not specifically depicted in FIG. 13, any
thermo-controllable units in the upper and/or the bottom arrays may
be sealed or unsealed. In addition, any thermo-controllable units
within the upper and/or the bottom arrays may be filled or
unfilled, with or without the reaction sample. Leaving one or more
thermo-controllable units unfilled enhances heat insulation because
air is a poor conductor of heat. Accordingly, it is preferable to
leave the adjacent thermo-controllable units empty so as to reduce
heat transfer from one thermo-controllable unit to the next unit.
It is also preferable to leave the entire upper or the bottom array
of thermo-controllable units empty in order to minimize heat
transfer from one layer to the next.
[0066] The subject array of thermo-controllable units is placed in
thermal contact with a heating element. This is achieved by
integrating the heating element as part of the chip, or by placing
the chip in contact with an external heating element.
[0067] The heating element can be any heating means, including
resistive heater or any other heat-generating device, which
converts electrical or electromagnetic energy into heat energy.
Preferred heating elements are micro-heaters compatible to the
arrayed thermo-controllable units in terms of size and
configuration. The micro-heater can be placed as a detachable unit
adjacent to, at the base and/or on top of the unit. Alternatively,
the micro-heater can be affixed to the interior or the exterior
surface of the thermo-controllable unit as an integral part of the
unit. The integral heating element may surround the micro well
wall, or located at the base of the thermo-controllable unit.
[0068] Micro-heaters are typically made of materials having high
thermal conductivity and chemical stability. Such materials include
but are not limited to metals such as chromium, platinum and gold,
and semi-conductors such as ceramic, silicon, and geranium. A
material particularly suitable for fabricating the micro-heaters is
indium tin oxide (ITO). ITO is a transparent ceramic material with
a very high electrical conductivity. Because ITO can be prepared in
bulk or in form of thin layer, it is particularly useful as either
an integral or an external heating element.
[0069] The integral micro-heater generally is made of resistive
heating material. Where the heating material is metal, it is
generally preferable to coat the surface with an insulating layer
to prevent corrosion and/or electrophoresis of the sample
components during operation with fluid samples. Coating is usually
not necessary in case of non-metal heating material. A variety of
protective coatings are available, including those made of, e.g.,
SiO.sub.2, Si.sub.3N.sub.4, and teflon. As is apparent to those
skilled in the art, certain heating materials can be deposited
directly onto the substrate of the thermo-controllable units, and
others may be deposited first onto an adhesion layer such as
titanium or glass that in turn attaches to the substrate of the
units.
[0070] The heating element is typically connected via electric
leads to a power source that provides voltage across the element
and effects subsequent heating of the thermo-controllable units.
The heating element may also be coupled to a temperature sensor
that monitors and regulates the temperature of the unit. The
temperature sensor may control the temperature and hence the
thermal profile of an array of thermo-controllable units. For
instance, FIG. 24 depicts an exemplary chip with multiple
temperature zones, in which each array of 96 thermo-controllable
units represents one temperature zone that is controlled by a
temperature sensor and exhibiting a thermal profile distinct from
the rest of the temperature zones. Dividing the chip into various
temperature zones provides additional flexibility for parallel
performance of chemical reactions that require different thermal
cycling profiles. Alternatively, the temperature sensor can be
coupled to individual thermo-controllable unit so that the
temperature of each unit can be independently controlled. The
temperature sensor may be included as a detachable unit located
adjacent to or at the base of the thermo-controllable unit. It can
also be integrated into the interior or the exterior surface of the
unit. Furthermore, the temperature sensor can be fabricated as an
integral part of the micro-heater.
[0071] The subject chips can be provided with an optical system
capable of detecting and/or monitoring the results or the progress
of chemical reactions taking place in the micro wells of the chips.
Such optical system achieves these functions by first optically
exciting the reactants, followed by collecting and analyzing the
optical signals from the reactants in the micro wells. The optical
system applicable for the present invention comprises three
elements, namely the optical excitation element, the optical
transmission element, and the photon-sensing element. The optical
system may also comprise, optionally, an optical selection
element.
[0072] The optical excitation element act as the source of
excitation beams used to optically excite the reactants contained
in the micro wells. This element encompasses a wide range of
optical sources that generate light beams of different wavelengths,
intensities and/or coherent properties. Representative examples of
such optical excitation sources include, but are not limited to,
lasers, light-emitting diodes (LED), ultra-violet light bulbs,
and/or white light sources.
[0073] The optical transmission element used in the present
invention serves two functions. First, it collects and/or directs
the optical excitation sources to the reactants inside the micro
wells of the chips. Second, it transmits and/or directs the optical
signals emitted from the reactants inside the micro wells of the
chips to the photon-sensing element. The optical transmission
element suitable for use in the present invention encompasses a
variety of optical devices that channel light from one location
point to another. Non-limiting examples of such optical
transmission devices include optical fibers, optical multiplexers
(MUX) and de-multiplexers (DE-MUX), diffraction gratings, arrayed
waveguide gratings (AWG), optical switches, mirrors, lenses,
collimators, and any other devices that guide the transmission of
light through proper refractive indices and geometries.
[0074] The photon-sensing element analyzes the spectra of the
optical signals coming from the reactants inside the micro wells.
Suitable photon-sensing element can detect the intensity of an
optical signal at a given wavelength, and preferably can
simultaneously measure the intensities of optical signals across a
range of wavelengths. Preferably the element may also provide
spectrum data analyses to show the spectrum peak wavelength,
spectrum peak width, and background spectrum noise measurements.
Representative examples of suitable photon-sensing element for the
present invention are avalanche photo diodes (APD), charge-coupled
devices (CCD), electron-multiplying charge-coupled device (EMCCD),
photo-multiplier tubes (PMT), photo-multiplier arrays, gate
sensitive FET's, nano-tube FET's, and P-I-N diode. As used herein,
CCD includes conventional CCD, electron-multiplying charge-coupled
device (EMCCD) and other forms of intensified CCD.
[0075] While the subject optical systems can be assembled using
many combinations of the various elements, a useful assembly for
analyzing the spectra of the excited reactants comprises an optical
transmission element and a photon-sensing element. Such assembly is
also referred to herein as "spectrum analyzer".
[0076] Where desired, the optical system of the present invention
can include an optical selection element. This element selects
and/or refines the optical properties of the excitation beams
before they reaches the reactants contained in the micro wells. The
optical selection element can also be employed to select and/or
refine the optical signals coming from the reactants in the
micro-wells before the signals reach the photon-sensing element.
Suitable optical selection element can select and modify a wide
range of optical properties, including but not limited to,
polarization, optical intensities, wavelengths, phase differences
among multiple optical beams, time delay among multiple optical
beams. Representative examples of such optical selection elements
are polarization filters, optical attenuators, wavelength filters
(low-pass, band-pass or high-pass), wave-plates and delay
lines.
[0077] The aforementioned optical elements can adopt a variety of
configurations. They can form integral parts of the subject chips
or remain as separate units. All of these elements are commercially
available. Accordingly, in one embodiment, the present invention
provides a chip in which the optical transmission and
photon-sensing elements are fabricated into the chip substrate. In
one aspect, the photon-sensing element is integrated into each
micro well on the chip that is to be monitored (see, e.g., FIGS. 1,
6A, and 11). In another aspect, more than one type of
photon-sensing element is integrated into the micro well to enhance
the detection capability or efficiency. In another aspect, the
photon-sensing element can be fabricated along the side or at the
base of the micro well, or as part of the cover of the micro well.
Photon-sensing elements suitable for such configuration include but
are not limited to avalanche photo diode, charge coupled devices
(including conventional CCD, electron-multiplying charge-coupled
device (EMCCD) and other forms of intensified CCD), gate sensitive
FET's, nano-tube FET's, P-I-N diode. Avalanche photo diode is
particularly preferred because it permits detections of a single
photon by amplifying the signal through an avalanche process of
electron transfer. These elements together with the supporting
circuitry can be fabricated as part of the subject chips using
standard IC processing techniques described herein or known in the
art.
[0078] In another embodiment, the present invention provides an
apparatus in which the chip and the optical systems remain as
separate units. One aspect of this embodiment encompasses an
apparatus for conducting a chemical or biological reaction
requiring cycling at least two temperature levels over a
multiple-cycle period. The apparatus comprises a chip of the
present invention, and an optical system that is operatively
coupled to the chip and that detects an optical signal coming from
the micro well. Preferably, the optical signals detected are
related to the amount of product of the chemical reaction taking
place in the micro well.
[0079] FIG. 16 illustrates an exemplary optical system of this
aspect. This system includes an array of optical transmission
element, namely the 1.times.L (where L is a positive integer)
arrayed waveguide 1606, that multiplexes up to L excitation beams
1610 into one optical beam 1612. The excitation beams may have the
same or different wavelengths ranging from, e.g., 200 nm to 1000
nm. A plurality of M (where M is a positive integer) arrayed
waveguides, each channeling a multiplexed beam, are connected to an
M.times.N optical switch 1608 via the respective optical fiber
1616. The M.times.N optical switch can direct M input excitation
beams from the arrayed waveguide 1606 to any one of its N output
ports. Each of the N output ports is operatively coupled to a micro
well through an optical fiber 1604. Suitable optical fibers
channeling the excitation beams to the micro well may include
multi-mode fibers (MMF) and single-mode fibers (SMF). Upon
excitation with the incident light beams, optical signals are
generated from the reactants inside the micro wells. These optical
signals are then collected via an optical collimator 1614 to a
1.times.P (where P is a positive integer) arrayed waveguide 1616
which de-multiplexes the optical signals. The de-multiplexed
optical signals are then transmitted to a spectrum analyzer, here a
charge-coupled device (CCD) 1618 (which is part of the spectrum
analyzer), for a spectrum analysis. CCDs having high number of
pixels are preferred as they provide a higher resolution of the
optical signals being examined.
[0080] Another exemplary optical system of the present invention is
depicted in FIG. 17. An array of fibers 1702 is employed to direct
a plurality of excitation beams of the same or different
wavelengths to a micro well 1704 on a chip. The fibers within the
array can be arranged in a circular configuration as shown in FIG.
17A or any other convenient configurations. The optical signals
coming from the reactants in the micro well is then collected and
transported by fibers 1706 to a spectrometer. The spectrometer
periodically and sequentially samples and analyzes the spectrum
outputs of the fibers 1706. The optimal sampling frequency can be
empirically determined. It may range from once every millisecond,
to once every 150 milliseconds, and to once every 1500
milliseconds. This configuration is particularly suited for a range
of spectroscopic applications because it permits the application of
a wide range of excitation wavelengths to a reaction sample being
examined. As such, the configuration supports analyses of
fluorescence, chemiluminescence, scintillation, bioluminescence,
and time-resolved applications without the need for frequent
re-alignment of the excitation sources that provide the appropriate
excitation wavelengths.
[0081] FIG. 19 depicts another exemplary optical system of the
present invention. In this system, optical fibers 1902 and beam
collimators 1903 connected to an excitation source 1901 are
employed to illuminate all the micro-wells on a chip 1904. The
excitation source can be high power tunable lasers or Xenon lamps.
The optical fibers 1902 are typically multi-mode fibers (MMF) of
one millimeter diameter. The collimated beams from the excitation
source preferably provide uniform energy distribution across all
the micro wells in the chip. The optical signals coming from the
micro wells on the silicon micro-plate are collimated by a lens
1905 and are passed through a tunable filter 1906 to an
electron-multiplying charge-coupled device (EMCCD) for spectrum
analysis. All of the elements including the optical fibers 1902,
collimating lens 1903 and 1905, silicon micro-plate 1904, tunable
filter 1906 and EMCCD 1907, are enclosed in a highly protected dark
housing 1908. This particular embodiment offers a low cost solution
for monitoring the progress and/or results of chemical reactions
taking place in micro wells fabricated on a chip.
Preparation of the Subject Chips
[0082] The chips of the present invention can be fabricated using
techniques well established in the Integrated Circuit (IC) and
Micro-Electro-Mechanical System (MEMS) industries. The fabrication
process typically proceeds with selecting a chip substrate,
followed by using appropriate IC processing methods and/or MEMS
micromachining techniques to construct and integrate various
components.
Chip Substrate:
[0083] Several factors apply to the selection of a suitable chip
substrate. First, the substrate must be a good thermal conductor. A
good thermal conductor generally has a thermal conductivity value
higher than 1 W/m.sup.-1K.sup.-1, preferably higher than 100
W/m.sup.-1K.sup.-1, more preferably higher than 140
W/m.sup.-1K.sup.-1. Whereas the material's thermal conductivity may
be 250 W/m.sup.-1K.sup.-1 or higher, it usually does not exceed 500
W/m.sup.-1K.sup.-1. Second, the substrate must be relatively inert
and chemically stable. Such substrate generally exhibits a low
level of propensity to react with the reaction samples employed in
the intended application. Moreover, the materials should also be
selected based upon the ability or feasibility to integrate the
thermal control elements onto or adjacent to them. A variety of
materials meet these criteria. Exemplary materials include but are
not limited to metalloids or semiconductors, such as silicon,
silicates, silicon nitride, silicon dioxide, gallium phosphide,
gallium arsenide, or any combinations thereof. Other possible
materials are glass, ceramics (including crystalline and
non-crystalline silicate, and non-silicate-based ceramics), metals
or alloys, composite polymers that contain dopants (e.g., aluminum
oxide to increase thermal conductivity), or any of a range of
plastics and organic polymeric materials available in the art.
Fabrication Process:
[0084] Fabrication of the subject chips can be performed according
to standard techniques of IC-processing and/or MEMS micromachining.
Typically, the subject chips are fabricated as multi-layer
structures. The process generally proceeds with constructing the
bottom layer. Then a combination of techniques including but not
limited to photolithography, chemical vapor or physical vapor
deposition, dry or wet etching are employed to build structures
located above or embedded therein. Vapor deposition, for example,
enables fabrication of an extremely thin and uniform coating onto
other materials, whereas etching allows for mass production of
larger chip structures. Other useful techniques such as ion
implantation, plasma ashing, bonding, and electroplating can also
be employed to improve the surface properties of the chips or to
integrate various components of the chips. The following details
the fabrication process with reference to the exemplary chip
designs depicted in the figures. The same general process and the
apparent variations thereof are applicable to fabricate any of the
subject chips described herein.
[0085] FIG. 5 is a cross-section of an exemplary chip design 501.
In this embodiment, the micro well 502 is embedded within a body
504 which is made up of first and second (or bottom and top) layers
of substrates 506 and 508, respectively. The process begins with
providing a first layer of substrate which is generally a heat
resistant material such as glass, Pyrex wafer, or any other
suitable materials described herein or known in the art. The next
step is to create the micro well 502 that forms the basis of the
thermo-controllable unit. The micro well is generally disposed
within the second layer 508 that is typically a silicon wafer. The
silicon wafer may go through several processing steps prior to
being attached to the first layer. For example, the silicon wafer
may be attached to a layer of photoresist to render the surface
more susceptible to chemical etching after exposure to UV light
during the process of photolithography. The layer of photoresist
defines, by precise alignment of the photo-mask, the size and
location of the micro well that is to be formed by a subsequent
etching step. The silicon wafer is then etched by a variety of
means known in the art to form the well cavity. A commonly
practiced etching technique involves the use of chemicals, e.g.,
potassium hydroxide (KOH), which removes the silicon wafer to form
the desired shape.
[0086] The heating element described herein can be deposited onto
the interior surface of the micro well (see, e.g., FIGS. 6A and
11). The micro-heaters, for example, may be arranged to surround
the micro well wall, or form the base of the micro well. The
micro-heater and the fluid contained in the well can be isolated
electrically and chemically from each other by an insulating or
protective coating. Coating is particularly preferable in case of
metal heating element that may be prone to corrosion and/or
electrophoresis of the sample components during operation with
fluid samples. A variety of protective coatings are available in
the art, including those made of, e.g., SiO.sub.2, Si.sub.3N.sub.4,
and Teflon. Where the heating element is indium tin oxide, it is
preferable to use glass (e.g. on borosilicate glass), quartz, or
the like material as the adhesion layer before depositing it into
the micro well.
[0087] Integrated circuitry that supports the operations of the
heating element and/or the temperature sensor can also be implanted
into the well or onto the exterior part of the silicon layer by a
suitable IC-processing technique described herein or known in the
art.
[0088] The second layer of silicon 508 in FIG. 5 or other suitable
substrate material can be attached to the first glass layer in one
of several ways. Anodic bonding can be used when the materials
employed are compatible with the bonding requirements.
Alternatively, a polymeric bonding compound such as
benzocyclobutene (BCB) (available from Dow Chemical) can be applied
to adhere one layer onto the next. In addition, the two layers of
substrates can be fused together by extensive heating under high
temperatures.
[0089] FIG. 6 and FIGS. 8-11 depict other exemplary chip designs
601 in which the first layer is made of silicon or the like
material. In FIGS. 6 and 11, the temperature sensors 604 and 1111,
heating elements 606 and 1006, and photo-sensing elements 608 and
1010 are fabricated in the first layer using methods described
above or other methods illustrated in the pending application Ser.
No. 10/691,036, the content of which is incorporated by reference
in its entirety.
[0090] To enhance the detection and sensing capabilities of the
chip, additional layers of sensing structures such as waveguides
610 are fabricated. The waveguides are constructed to channel light
beams emitted from one or multiple micro wells through a side wall
of the micro well 610. While it may be preferable to couple one
waveguide to a single micro well to effect separate detection of
light signals emitted from individual wells, channeling signals
from multiple wells are possible by adjusting the excitation light
beam. For instance, the incoming light can be synchronized in or
out of phase with light signals collected from other waveguides
such that multiple pulses of light beams of known wavelengths and
intensities arrive at different micro wells within predetermined
time frames. The sensor reading associated with that particular
light pulse is then monitored with appropriate post processing. The
materials with which the waveguides are fabricated are determined
by the wavelength of the light being transmitted. Silicon dioxide
is suitable for transmitting light beams of a wide spectrum of
visible wavelengths. Silicon and polysilicon are applicable for
guiding infra-red wavelengths. Those skilled in the art will know
of other materials suitable for constructing waveguide. To achieve
the desired polarization states, waveguides with appropriate
integral gratings can be constructed using standard MEMS
micromachining techniques.
[0091] The chip depicted in FIG. 6A also contains a top layer
micro-heater. The top layer heater provides an additional source of
heat energy to effect a rapid thermal cycling. It may also serve as
a physical barrier to prevent evaporation of the reaction reagents
applied to the micro well. To further minimize evaporation, the top
layer heater can be maintained at a higher temperature, usually a
few Celsius degrees higher relative to the bottom heater. The type
of heater to be placed on the upper surface will depend on the
intended use of the chip. Indium tin oxide heater is generally
preferred because it is transparent to visible light. When
deposited on glass and applied to the top of the chip, light
emitted from the micro well can still pass through and be detected
by a photon-sensing element.
[0092] Once the micro wells of the subject chips are fabricated,
their surface properties can be improved to suit the particular
application. Where large surface area is desired, the wall of the
micro well may be further etched by, e.g., a plasma etcher to
obtain very fine dendrites of silicon, commonly referred to as
"black silicon". The presence of black silicon can dramatically
increase the effective heating surface area. The black silicon
fabricated at the base of the micro well may also serve as an
anchor for photon-sensing devices, temperature sensors and other
control elements.
[0093] As discussed in the sections above, a micro well of high
inner surface to volume ratio may be coated with materials to
reduce the possibility that the reactants contained therein may
interact with the inner surfaces of the well. The choice of methods
for applying the coating materials will depend on the type of
coating materials that is used. In general, coating is carried out
by directly applying the materials to the micro well followed by
washing the excessive unbound coating material. Certain coating
materials can be cross-linked to the surface via extensive heating,
radiation, and by chemical reactions. Those skilled in the art will
know of other suitable means for coating a micro well fabricated on
chip, or will be able to ascertain such, without undue
experimentation.
[0094] The surface of the micro well can further be altered to
create adsorption sites for reaction reagents. One skilled in the
art will appreciate that there are many ways of creating adsorption
sites to immobilize chemical or biological reactants. For instance,
a wealth of techniques are available for directly immobilizing
nucleic acids and amino acids on a chip, anchoring them to a linker
moiety, or tethering them to an immobilized moiety, via either
covalent or non-covalent bonds (see, e.g., Methods Mol. Biol. Vol.
20 (1993), Beier et al., Nucleic Acids Res. 27:1970-1-977 (1999),
Joos et al., Anal. Chem. 247:96-101 (1997), Guschin et al., Anal.
Biochem. 250:203-211 (1997)).
[0095] The subject chips can be further modified to contain one or
more grooves on the top, or at the bottom side of the chip (see,
e.g., FIGS. 6A and 9). Grooves are generally fabricated by etching
the bottom side silicon wafer. Back-side etching can be carried out
before or after formation of the micro well.
Sealing Process
[0096] In most of the applications, sealing the micro wells is
desirable to prevent evaporation of liquids and thus maintains the
preferred reaction concentrations throughout the thermal cycling.
Accordingly, the present invention provides a technique for sealing
an array of micro wells. The design of the subject sealing
technique takes several factors into consideration. First, the
technique should be amenable to high throughout processing of a
large quantity of micro wells. Second, the method should permit
selective sealing of individual micro wells. As such, the method
can yield chips comprising open micro wells interspersed among
sealed micro wells in any desired pattern or format. As mentioned
above, chips having both open and sealed micro wells are
particularly desirable. The open and/or unfilled wells not only
allow passive dissipation of heat, but also reduce heat transfer
between the neighboring micro wells.
[0097] A preferred method of sealing an array of micro wells
containing at least one open well. The method comprises the steps
of (a) applying a radiation-curable adhesive along peripheral
dimensions defining the open surface of the at least one open micro
well; (b) placing a cover to encompass the peripheral dimensions
that define the open surface of the at least one open micro well
that is to be sealed; and (c) exposing the array to a radiation
beam to effect the sealing.
[0098] As used herein, "radiation-curable adhesive" refers to any
composition that cures and bonds to the adhering surface upon
exposure to a radiation beam without the need of extensive heating.
"Radiation beam" refers to electromagnetic waves of energy
including, in an ascending order of frequency, infrared radiation,
visible light, ultraviolet (VU) light, X-rays, and gamma rays. A
vast number of radiation-curable adhesive are commercially
available (see, e.g., a list of companies selling radiation-curable
adhesive and radiation systems from ThomasNet.RTM.'s worldwide web
site). They include a diversity of acrylics, acrylates,
polyurethanes (PUR), polyesters, vinyl, vinyl esters, and a vast
number of epoxies that are curable by radiation beams at various
frequencies. These and other radiation-curable materials are
supplied commercially in form of liquid, or solid such as paste,
powder, resin, and tape.
[0099] The choice of radiation-curable adhesive will dependent on
the materials made up the surfaces to be adhered. The
aforementioned classes of adhesive are suited for adhering the chip
substrate to the cover which can be made of a range of materials.
For instance, acrylics and epoxies are applicable for
radiation-sealing any two surfaces, made of any one of the
materials selected from glass, ceramics, metalloids, semiconductors
(e.g., silicon, silicates, silicon nitride, silicon dioxide,
quartz, and gallium arsenide), plastics, and other organic
polymeric materials. Radiation-curable materials exhibiting the
properties of low use temperature and rapid curing time are
particularly desirable for sealing the subject chips. These
materials allow for a rapid sealing to avoid radiation damages to
the chemical or biological reagents contained in the chips.
[0100] The radiation-curable adhesive can be applied by any
mechanical means along the peripheral dimensions that define the
open surface of a micro well. The "peripheral dimensions" can be
the boundaries on the chip substrate or on the cover. In either
case, the peripheral dimensions become bonded to the respective
adhering surface, the substrate or the cover, upon curing the
adhesive. The radiation-curable adhesive can be smeared, printed,
dispensed, or sprayed onto the peripheral dimensions using any
suitable tools. Preferred mechanical means yields a uniform layer
of adhesive on the peripheral dimensions. One way to provide a
uniform distribution is to apply the adhesive directly onto the
peripheral dimensions of an open well using a squeegee over a
meshed screen mask (see, e.g., FIG. 25). Alternatively, the
radiation-curable adhesive can be applied directly onto the cover
that has been marked with the peripheral dimensions using the
meshed screen mask. A uniform layer of adhesive is achieved upon
removal of the mask.
[0101] Upon application of the radiation-curable adhesive, a cover
is placed on the micro well to encompass the peripheral dimensions
that define the open surface of the well. Suitable covers are
generally made of materials that permit passage of a radiation
beam. Preferred covers are fabricated with transparent materials
such as glass, quartz, plastic, any suitable organic polymeric
materials known to those skilled in the art, or any combinations
thereof.
[0102] Sealing a covered micro well is carried out by exposing the
well to a radiation beam. Depending on the type of adhesive
selected, the radiation beam may come from a conventional
incandescent source, a laser, a laser diode, UV-bulb, an X-ray
machine or gamma-ray machine, or the like. Where desired, radiation
beam from the radiation source is permitted to reach only selected
locations on the micro well array so that only certain selected
wells are to be sealed. A selective sealing is often achieved by
using a photo-mask patterned with the locations of the micro wells.
The photo-mask is provided with transparent locations and opaque
locations that correspond to the micro wells that are to be sealed
and those that are to remain open, respectively. The radiation beam
passes freely through the transparent regions but is reflected from
or absorbed by the opaque regions. Therefore, only selected micro
wells are exposed to light and hence sealed by curing the adhesive.
In one embodiment, the photo-mask is patterned such that no two
adjoining open micro wells are to be sealed. In another embodiment,
the photo-mask is patterned such that the resulting micro well
array contains alternating sealed and unsealed wells. One skilled
in the art can fashion an unlimited number of photo-masks with any
patterns to yield chips containing open and sealed micro wells in
any format. Methods for manufacturing such photo-masks are well
established in the art and hence are not detailed herein.
Uses of the Subject Chip and Other Devices of the Present
Invention
[0103] The subject chips have a wide variety of uses in chemical
and biological applications where controllable temperatures are
desired.
[0104] In one embodiment, the subject chips can be used to vary
and/or maintain temperature of a reaction sample. Varying and/or
maintaining temperature of a reaction sample are required in a wide
range of circumstances including but not limited to discerning
protein-protein interaction, examining DNA or RNA hybridization,
and performing enzymatic reaction. The method involves placing the
reaction sample into a micro well fabricated in a chip that is in
thermal contact with a heating element, and applying a voltage to
the heating element.
[0105] In another embodiment, the subject chips are used for
conducting a chemical reaction that involves a plurality of
reaction samples and requires cycling at least two temperature
levels. The process involves (a) providing a chip comprising an
array of thermo-controllable units as described herein; (b) placing
the plurality of reaction samples into the thermo-controllable
units of the chip; and (c) controlling the heating element to
effect cycling at least two temperature levels.
[0106] As used herein, the term "chemical reaction" refers to any
process involving a change in chemical properties of a substance.
Such process includes a vast diversity of reactions involving
biological molecules such as proteins, glycoproteins, nucleic
acids, and lipids, or inorganic chemicals, or any combinations
thereof. The chemical reaction may also involve interactions
between nucleic acid molecules, between proteins, between nucleic
acid and protein, between protein and small molecules. Where the
process is catalyzed by an enzyme, it is also referred to as
"enzymatic reaction."
[0107] The subject chips and other apparatus are particularly
useful in conducting enzymatic reactions because most enzymes
function under only certain temperatures. Representative enzymatic
reactions that are particularly temperature dependent include but
are not limited to nucleic acid amplification, quantitative
polymerase chain reaction (qPCR), nucleic acid sequencing, reverse
transcription, and nucleic acid ligation.
[0108] Practicing the subject method generally proceeds with
placing the reaction sample into a micro well of the subject chip
that is in thermal contact with a heating element. Where desired,
the reaction sample can be applied by a dispensing system
operatively coupled to the subject chip. A variety of dispensing
instruments, ranging from manually operated pipettes to automated
robot systems are available in the art. Preferred dispensing
instruments are compatible to the particular format (e.g. 96-well)
of the subjects chip.
[0109] To prevent evaporation of aqueous reaction samples, the
samples can be applied to the micro well at or around dew point. As
used herein, "dew point" refers to a temperature range where the
droplet size does not change significantly. At dew point, an
equilibrium is reached between the rate of evaporation of water
from the sample droplet and the rate of condensation of water onto
the droplet from the moist air overlying the chip. When this
equilibrium is realized, the air is said to be saturated with
respect to the planar surface of the chip. At one atmospheric
pressure, the dew point is about 14.degree. C. Accordingly,
dispensing aqueous reaction samples is preferably carried out at a
temperature no more than about 1.degree. C. to about 5.degree. C.
degrees above dew point. As is apparent to one skilled in the art,
dew point temperature increases as the external pressure increases.
Therefore, where desired, one may dispense the reaction samples in
a pressured environment to prevent evaporation.
[0110] In practice, controlling the heating element and hence the
temperature of the reaction sample, is effected by processing a
predetermined algorithm stored on a computer readable medium
operatively linked to the heating element. In other aspects, the
controlling step may involve processing sensor signals retrieved
from a temperature sensor element that is operatively linked to a
thermo-controllable unit based on protocols stored on a computer
readable medium. This can be achieved by employing conventional
electronics components for temperature control that may process
either analog or digital signals. Preferably, the electronics
components are run on a feedback control circuitry. They can
control the temperature of one unit, but more often the temperature
of multiple thermo-controllable units that collectively form one
temperature zone. Where desired, the chemical reactions can take
place in different thermo-controllable units located in different
temperature zones. In certain embodiments, the temperatures of the
different zones are separately controlled. The thermal cycling
profile and duration will depend on the particular application in
which the subject chip is to be employed.
Nucleic Acid Amplification:
[0111] The chips of the present invention provide a cost-effective
means for amplifying nucleic acids. Unlike the conventional thermal
cyclers, the subject chips are highly miniaturized, capable of
performing rapid amplification of a vast number of target nucleic
acids in small volume, and under independent thermal protocols.
[0112] As used herein, "nucleic acid amplification" refers to an
enzymatic reaction in which the target nucleic acid is increased in
copy number. Such increase may occur in a linear or in an
exponential manner. Amplification may be carried out by natural or
recombinant DNA polymerases such as Taq polymerase, Pfu polymerase,
T7 DNA polymerase, Klenow fragment of E. coli DNA polymerase,
and/or RNA polymerases such as reverse transcriptase.
[0113] A preferred amplification method is polymerase chain
reaction (PCR). General procedures for PCR are taught in U.S. Pat.
No. 4,683,195 (Mullis) and U.S. Pat. No. 4,683,202 (Mullis et al.).
Briefly, amplification of nucleic acids by PCR involves repeated
cycles of heat-denaturing the DNA, annealing two primers to
sequences that flank the target nucleic acid segment to be
amplified, and extending the annealed primers with a polymerase.
The primers hybridize to opposite strands of the target nucleic
acid and are oriented so that the synthesis by the polymerase
proceeds across the segment between the primers, effectively
doubling the amount of the target segment. Moreover, because the
extension products are also complementary to and capable of binding
primers, each successive cycle essentially doubles the amount of
target nucleic acids synthesized in the previous cycle. This
results in exponential accumulation of the specific target nucleic
acids at approximately a rate of 2.sup.n, where n is the number of
cycles.
[0114] A typical conventional PCR thermal cycling protocol
comprises 30 cycles of (a) denaturation at a range of 90.degree. C.
to 95.degree. C. for 0.5 to 1 minute, (b) annealing at a
temperature ranging from 55.degree. C. to 65.degree. C. for 1 to 2
minutes, and (c) extension at 68.degree. C. to 75.degree. C. for at
least 1 minute with the final cycle extended to 10 minutes. With
the subject chips, the thermal cycling time can be drastically
reduced because of, partly, the small reaction volume, the small
heating mass, and the design of effective heat dissipation
features.
[0115] A variant of the conventional PCR is a reaction termed "Hot
Start PCR". Hot Start PCR techniques focus on the inhibition of
polymerase activity during reaction preparation. By limiting
polymerase activity prior to PCR cycling, non-specific
amplification is reduced and the target yield is increased. Common
methods for Hot Start PCR include chemical modifications to the
polymerase (see, e.g., U.S. Pat. No. 5,773,258), inhibition of the
polymerase by a polymerase-specific antibody (see, e.g., U.S. Pat.
No. 5,338,671), and introduction of physical barriers in the
reaction site to sequester the polymerase before the thermal
cycling takes place (e.g., wax-barrier methods). The reagents
necessary for performing Hot Start PCR are conveniently packaged in
kits that are commercially available (see, e.g., Sigma's JumpStart
Kit).
[0116] Another variant of the conventional PCR that can be
performed with the subject chips is "nested PCR" using nested
primers. The method is preferred when the amount of target nucleic
acid in a sample is extremely limited for example, where archival,
forensic samples are used. In performing nested PCR, the nucleic
acid is first amplified with an outer set of primers capable of
hybridizing to the sequences flanking a larger segment of the
target nucleic acid. This amplification reaction is followed by a
second round of amplification cycles using an inner set of primers
that hybridizes to target sequences within the large segment.
[0117] The subject chips can be employed in reverse transcription
PCR reaction (RT-PCR). RT-PCR is another variation of the
conventional PCR, in which a reverse transcriptase first coverts
RNA molecules to double stranded cDNA molecules, which are then
employed as the template for subsequent amplification in the
polymerase chain reaction. In carrying out RT-PCR, the reverse
transcriptase is generally added to the reaction sample after the
target nucleic acids are heat denatured. The reaction is then
maintained at a suitable temperature (e.g., 30-45.degree. C.) for a
sufficient amount of time (e.g., 5-60 minutes) to generate the cDNA
template before the scheduled cycles of amplification take place.
Such reaction is particularly useful for detecting the biological
entity whose genetic information is stored in RNA molecules.
Non-limiting examples of this category of biological entities
include RNA viruses such as HIV and hepatitis-causing viruses.
Another important application of RT-PCR embodied by the present
invention is the simultaneous quantification of biological entities
based on the mRNA level detected in the test sample. One of skill
in the art will appreciate that if a quantitative result is
desired, caution must be taken to use a method that maintains or
controls for the relative copies of the amplified nucleic
acids.
[0118] Methods of "quantitative" amplification of nucleic acids are
well known to those of skill in the art. For example, quantitative
PCR (qPCR) can involve simultaneously co-amplifying a known
quantity of a control sequence using the same primers. This
provides an internal standard that may be used to calibrate the PCR
reaction. Other ways of performing qPCR are available in the art
and are detailed in the section "Detection of Amplified Target
Nucleic Acids" below.
[0119] The subject chips can also be employed to form ligase chain
polymerase chain reaction (LCR-PCR). The method involves ligating
the target nucleic acids to a set of primer pairs, each having a
target-specific portion and a short anchor sequence unrelated to
the target sequences. A second set of primers containing the anchor
sequence is then used to amplify the target sequences linked with
the first set of primers. Procedures for conducting LCR-PCR are
well known to artisans in the field, and hence are not detailed
herein (see, e.g., U.S. Pat. No. 5,494,810).
[0120] Nucleic acid amplification is generally performed with the
use of amplification reagents. Amplification reagents typically
include enzymes, aqueous buffers, salts, primers, target nucleic
acid, and nucleoside triphosphates. Depending upon the context,
amplification reagents can be either a complete or incomplete
amplification reaction mixture.
[0121] The choice of primers for use in nucleic acid amplification
will depend on the target nucleic acid sequence. Primers used in
the present invention are generally oligonucleotides, usually
deoxyribonucleotides several nucleotides in length, that can be
extended in a template-specific manner by the polymerase chain
reaction. The design of suitable primers for amplifying a target
nucleic acid is within the skill of practitioners in the art. In
general, the following factors are considered in primer design: a)
each individual primer of a pair preferably does not
self-hybridize; b) the individual pairs preferably do not
cross-hybridize; and c) the selected pair must have the appropriate
length and sequence homology in order to anneal to two distinct
regions flanking the nucleic acid segment to be amplified. However,
not every nucleotide of the primer must anneal to the template for
extension to occur. The primer sequence need not reflect the exact
sequence of the target nucleic acid. For example, a
non-complementary nucleotide fragment may be attached to the 5' end
of the primer with the remainder of the primer sequence being
complementary to the target. Alternatively, non-complementary bases
can be interspersed into the primer, provided that the primer
sequence has sufficient complementarily with the target for
annealing to occur and allow synthesis of a complementary nucleic
acid strand.
[0122] For a convenient detection of the amplified nucleotide acids
resulting from PCR or any other nucleic acid amplification
reactions described above or known in the art, primers may be
conjugated to a detectable label. Detectable labels suitable for
use in the present invention include any composition detectable by
spectroscopic, photochemical, biochemical, immunochemical,
electrical, optical or chemical means. A wide variety of
appropriate detectable labels are known in the art, which include
luminescent labels, enzymatic or other ligands. In preferred
embodiments, one will likely desire to employ a fluorescent label
or an enzyme tag, such as digoxigenin, .beta.-galactosidase,
urease, alkaline phosphatase or peroxidase, avidin/biotin
complex.
[0123] The labels may be incorporated by any of a number of means
well known to those of skill in the art. In one aspect, the label
is simultaneously incorporated during the amplification step. Thus,
for example, PCR with labeled primers or labeled nucleotides can
provide a labeled amplification product. In a separate aspect,
transcription reaction in which RNA is converted into DNA, using a
labeled nucleotide (e.g. fluorescein-labeled UTP and/or CTP) or a
labeled primer, incorporates a detectable label into the
transcribed nucleic acids.
[0124] The primer pairs used in this invention can be obtained by
chemical synthesis, recombinant cloning, or a combination thereof.
Methods of chemical polynucleotide synthesis are well known in the
art and need not be described in detail herein. One of skill in the
art can use the target sequence to obtain a desired primer pairs by
employing a DNA synthesizer or ordering from a commercial
service.
[0125] Nucleic acid amplification requires a target nucleic acid in
a buffer compatible with the enzymes used to amplify the target.
The target nucleic acid used for this invention encompasses any
reaction samples suspected to contain the target sequence. It is
not intended to be limited as regards to the source of the reaction
sample or the manner in which it is made. Generally, the test
sample can be biological and/or environmental samples. Biological
samples may be derived from human, other animals, or plants, body
fluid, solid tissue samples, tissue cultures or cells derived
therefrom and the progeny thereof, sections or smears prepared from
any of these sources, or any other samples suspected to contain the
target nucleic acids. Preferred biological samples are body fluids
including but not limited to blood, urine, spinal fluid,
cerebrospinal fluid, sinovial fluid, ammoniac fluid, semen, and
saliva. Other types of biological sample may include food products
and ingredients such as vegetables, dairy items, meat, meat
by-products, and waste. Environmental samples are derived from
environmental material including but not limited to soil, water,
sewage, cosmetic, agricultural and industrial samples.
[0126] Preparation of nucleic acids contained in the test sample
can be carried out according to standard methods in the art or
procedures described. Briefly, DNA and RNA can be isolated using
various lytic enzymes or chemical solutions according to the
procedures set forth in Sambrook et al. ("Molecular Cloning: A
Laboratory Manual"), or extracted by nucleic acid binding resins
following the accompanying instructions provided by manufacturers'
instructions.
[0127] The nucleic acid in the reaction sample can be cDNA, genomic
DNA or viral DNA. However, the present invention can also be
practiced with other nucleic acids, such as mRNA, ribosomal RNA,
viral RNA. These nucleic acids may exist in a variety of
topologies. For example, the nucleic acids may be single stranded,
double-stranded, circular, linear or in form of concatamers. Those
of skill in the art will recognize that whatever the nature of the
nucleic acid, it can be amplified merely by making appropriate and
well recognized modifications to the method being used.
Detection of Amplified Target Nucleic Acid:
[0128] Amplified nucleic acids present in the subject chips may be
detected by a range of methods including but not limited to (a)
forming a detectable complex by, e.g., binding the amplified
product with a detectable label; and (b) electrophoretically
resolve the amplified product from reactants and other components
of the amplification reaction.
[0129] In certain embodiments, the amplified products are directly
visualized with detectable label such as a fluorescent DNA-binding
dye. Because the amount of the dye intercalated into the
double-stranded DNA molecules is typically proportional to the
amount of the amplified DNA products, one can conveniently
determine the amount of the amplified products by quantifying the
fluorescence of the intercalated dye using the optical systems of
the present invention or other suitable instrument in the art.
DNA-binding dye suitable for this application include SYBR green,
SYBR blue, DAPI, propidiuni iodine, Hoeste, SYBR gold, ethidium
bromide, acridines, proflavine, acridine orange, acriflavine,
fluorcoumanin, ellipticine, daunomycin, chloroquine, distamycin D,
chromomycin, homidium, mithramycin, ruthenium polypyridyls,
anthramycin, and the like.
[0130] In addition to various kinds of fluorescent DNA-binding dye,
other luminescent labels such as sequence specific probes can be
employed in the amplification reaction to facilitate the detection
and quantification of the amplified product. Probe based
quantitative amplification relies on the sequence-specific
detection of a desired amplified product. Unlike the dye-based
quantitative methods, it utilizes a luminescent, target-specific
probe (e.g., TaqMan.RTM. probes) resulting in increased specificity
and sensitivity. Methods for performing probe-based quantitative
amplification are well established in the art and are taught in
U.S. Pat. No. 5,210,015.
[0131] The subject chips and the associated optical systems are
particularly suited for conducting quantitative nucleic acid
amplification. Accordingly, the present invention provides a method
for monitoring the formation of a nucleic acid amplification
reaction product, preferably in real time. In certain preferred
embodiments, the amplified nucleic acids contained are directly
monitored by the photon-sensing elements integrated into the chips.
The photon-sensing element registers the intensities of the optical
signals that are reflective of the amount of the amplified nucleic
acids at any time being examined during the amplification reaction.
The optical signals may be any kind of luminescent signals emitted
upon exciting the labeled reactants with appropriate incident
beams.
[0132] In another preferred embodiment, the amplified nucleic acids
in the subject chips are detected by the subject optical systems
operatively coupled to the chips. The optical systems are capable
of transmitting appropriate excitation beams to the reactants in
the amplification reactions, collecting and analyzing the emitted
optical signals from the reactants. Preferably, the optical signals
detected are indicative of the amount of amplified nucleic acid in
the amplification reaction over a multiple-cycle period. In certain
aspects, the optical system transmits excitation beams into the
wells containing the reaction samples at a plurality of times
during the amplification, and monitors the optical signals coming
from the micro wells at each of the plurality of times. By
analyzing the relative intensities of the optical signals,
preferably over a multiple-cycle period, one can monitor
quantitatively the progression of the amplification reaction.
Typically, the optical signals being monitored are luminescent
signals. In certain preferred aspects, detecting and/or monitoring
the amplification products are performed without opening the micro
well once the amplification is initiated.
Uses of Nucleic Acid Amplification and Detection Techniques of the
Present Invention:
[0133] The subject methods of amplifying and detecting a target
nucleic acid have broad spectrum of utility in, e.g. drug
screening, disease diagnosis, phylogenetic classification,
genotyping individuals, parental and forensic identification.
[0134] At a more fundamental level, amplification and detection of
the target nucleic acids may be used in identification and
quantification of differential gene expression between diseased and
normal tissues, among different types of tissues and cells, amongst
cells at different developmental stages or at different cell-cycle
points, and amongst cells that are subjected to various
environmental stimuli or lead drugs.
Other Chemical and Biological Applications:
[0135] The subject chips and other devices find utility in many
other chemical and biological applications where controllable
temperatures are desired. Such applications include a vast
diversity of reactions such as redox reactions, hydrolysis,
phosphorylation, and polymerization. Additional applications are
directed to discerning interactions involving biological molecules
such as proteins, glycoproteins, nucleic acids, and lipids, as well
as inorganic chemicals, or any combinations thereof. The chemical
reaction may also involve interactions between nucleic acid
molecules, between nucleic acid and protein, between protein and
small molecules The chemical reaction may take place outside a cell
or inside a cell that is introduced into a micro well of the
subject chip.
[0136] Of particular significance is the application in detecting
the presence of a specific protein-protein interaction. Such
application generally employs a proteinaceous probe and a target
protein placed in a micro well in the subject chip.
[0137] In one aspect of this embodiment, the protein-protein
interaction is between a target protein (i.e. an antigen) and an
antibody specific for that target. In another aspect, the
protein-protein interaction is between a cell surface receptor and
its corresponding ligand. In yet another aspect, the
protein-protein interaction involves a cell surface receptor and an
immunoliposome or an immunotoxin; in other aspects, the
protein-protein interaction may involve a cytosolic protein, a
nuclear protein, a chaperon protein, or proteins anchored on other
intracellular membranous structures.
[0138] The terms "membrane", "cytosolic", "nuclear" and "secreted"
as applied to cellular proteins specify the extracellular and/or
subcellular location in which the cellular protein is mostly,
predominantly, or preferentially localized.
[0139] "Cell surface receptors" represent a subset of membrane
proteins, capable of binding to their respective ligands. Cell
surface receptors are molecules anchored on or inserted into the
cell plasma membrane. They constitute a large family of proteins,
glycoproteins, polysaccharides and lipids, which serve not only as
structural constituents of the plasma membrane, but also as
regulatory elements governing a variety of biological
functions.
[0140] The reaction is typically performed by contacting the
proteinaceous probe with a target protein under conditions that
will allow a complex to form between the probe and the target. The
conditions such as the reaction temperature, the duration of the
reaction, the buffer conditions and etc., will depend on the
particular interaction that is being investigated. In general, it
is preferable to perform the reactions under physiologically
relevant temperature and buffer conditions. Physiologically
relevant temperatures range from approximately room temperature to
approximately 37.degree. C. This can be achieved by adjusting the
heating element of the subject chips. Typically, a physiological
buffer contains a physiological concentration of salt and at
adjusted to a neutral pH ranging from about 6.5 to about 7.8, and
preferably from about 7.0 to about 7.5. A variety of physiological
buffers is listed in Sambrook et al. (1989) supra and hence is not
detailed herein.
[0141] The formation of the complex can be detected directly or
indirectly according standard procedures in the art or by methods
describe herein. In the direct detection method, the probes are
supplied with a detectable label and when a complex is formed, the
probes emitted an optical signal distinct from that of the
unreacted probes. A desirable label generally does not interfere
with target binding or the stability of the resulting target-probe
complex. As described above, a wide variety of labels suitable for
such application are known in the art, most of which are
luminescent probes. The amount of probe-target complexes formed
during the binding reaction can be quantified by standard
quantitative assays, or the quantitative methods using the optical
systems described above.
[0142] Further illustration of the design and use of the chips
according to this invention is provided in the Example section
below. The example is provided as a guide to a practitioner of
ordinary skill in the art, and is not meant to be limiting in any
way.
EXAMPLE 1
[0143] Amplification of a target nucleic acid, namely a fragment of
the G6PDH gene, is performed using a chip of the present invention.
The reaction mixture containes G6PDH template, a pair of upstream
and downstream primers specific for the template, dNTPs, and DNA
polymerase.
[0144] The amplified products are detected with SYBR Green I that
binds preferentially to double-stranded DNA molecules (see, FIG.
20). FIG. 21 depicts the SYBR Green staining of DNA molecules
appeared at the three different thermal stages of one PCR cycle. As
is shown in FIG. 21, staining of the DNA is most intense at the
annealing step (e.g., at 45.degree. C.) because most of the DNA
molecules assume a double helical structure. By contrast, very
little SYBR Green staining is detected at the denaturing step where
the temperature is raised to e.g., 95.degree. C. At about
72.degree. C. where primer-directed extension is taking place, a
moderate amount of staining is detected. The amount of SYBR Green
stain detected throughout one complete thermal cycle is quantified
as shown in FIG. 22.
* * * * *